Novel feeding processes for fermentation

ABSTRACT

Embodiments of the present invention generally relate to novel fed-batch fermentations wherein processes of DO-stat and pH-stat are combined for nutrient feeding control.

FIELD OF THE INVENTION

[0001] Embodiments of the present invention generally relate to novelprocesses for fermentation.

BACKGROUND OF THE INVENTION

[0002] The Pharmaceutical Industry has produced compounds of interestthrough fermentative production for a very long time. Generally, in afermentation production facility, a compound (or compounds) of interest,such as certain organic compounds, proteins, carbohydrates, and the likecan be produced in large quantities by culturing cells (often referredto as “host cell(s)”) in a liquid nutrient solution called medium. Thesehost cells produce the compound(s) of interest either naturally orthrough genetic engineering or recombinant DNA technology.

[0003] In an embodiment, to culture the host cells, typically, the cellsare submerged in a tank (often referred to as fermentor or bioreactor)of varying size containing a nutrient medium. This nutrient mediumallows the cells to grow, multiply, and synthesize the compound(s) ofinterest. This process is often referred to as fermentation or cellculture. Harvesting the compound(s) of interest often requiresextracting the compound(s) directly from the cells or from thesupernatant.

[0004] Two commonly used fermentation systems are the continuous culturesystem and the fed-batch culture system, according to U.S. Pat. No.5,639,658 to Drobish et al. The continuous culture system is typicallyused to extend the growth phase of the culture cells over long periodsof time by providing fresh medium to the cells while simultaneouslyremoving spent medium and cells from the fermentor. Such a culturingsystem serves to maintain optimal culturing conditions for certain hostcell types and compounds of interest. The fed-batch fermentation systemis generally defined as batch culture systems wherein fresh nutrientsand/or other additives such as precursors to products are added asdemanded by the fermentation process but no medium is withdrawn. Thereare three primary types of medium: chemically defined media,semi-defined media, and rich complex media.

[0005] A chemically defined medium as is illustrated in U.S. patentapplication Ser. No. 09/982,474, published on Apr. 4, 2002, is a mediumessentially composed of chemically defined constituents. A semi-definedmedium refers to a chemically defined medium supplemented with a smallamount of complex nitrogen and/or carbon source(s), an amount as definedbelow, which typically is not sufficient to maintain growth of themicro-organism and/or the guarantee formation of a sufficient amount ofbiomass.

[0006] A rich complex medium is typically defined as a medium comprisinga complex nitrogen and/or carbon source, such as soybean meal, cottonseed meal, corn steep liquor, yeast extract, casein hydrolysate,molasses, and the like. Likewise, a complex medium is a complete ornearly complete nutrient source for the microorganism. Rich complexmedia, in embodiments contain a carbon and a nitrogen source as well asvitamins, trace metals and minerals.

[0007] There are two primary types of fermentation: solid-statefermentation and aqueous fermentation. Solid-state fermentation includesthe steps of cultivation of media, inoculation of the media withmicroorganisms, cultivation of the multi-organisms, extraction ofbiological products from the cultivated microorganisms and treatment ofthe waste materials from the culture. Solid-state fermentation isdisclosed in U.S. Pat. No. 6,197,573 to Suryanarayan et al. Aqueousstate fermentation is as disclosed below.

[0008] As is disclosed in U.S. Pat. No. 5,595,905 to Bishop et al,during a fermentation process, the bacteria or yeast growing in thefermentation broth consume nutrients at a variable rate. This rate isoften related to such factors as the microorganism density and rate ofgrowth. It is common in the fed-batch fermentation that the rate ofconsumption of nutrients will increase exponentially until an upperlimit is reached for the fermentation that is often determined by thesize of the fermentor or amount of nutrient and dissolved oxygenavailable in the medium.

[0009] As is disclosed in the prior art, it is desirable to maintain anadequate or sufficient concentration of nutrient in the medium. When thenutrient concentration is too high, either undesirable by-products,usually acetic acid, lactic acid or ethanol are produced, or growthinhibition is observed due to nutrient toxicity at higher concentrations^([4]). When the nutrient concentration is too low the microorganismgrowth rate is restricted. Accordingly, the art field has strived tocontrol the nutrient concentration in the medium. This is often involveddifferent feeding patterns or measurements.

[0010] For example, in U.S. Pat. No. 5,595,905, patentees disclosetaking samples from a culture medium, analyzing those samples, and basedupon the analysis adding further nutrients to the water in an attempt tokeep the nutrient concentration at constant in the culture. The '905patent discloses using a computer to assist in monitoring the nutrientconcentration.

[0011] U.S. Pat. No. 6, 284, 453 discloses general approaches toimproving product formation that include, 1) using the best growthmedium (carbon source, nitrogen source, precursors, and nutrients suchas vitamins and minerals); 2) using the optimal pH, redox potential,agitation rate, aeration rate, ionic strength, osmotic pressure, wateractivity, hydrostatic pressure, and/or the like; 3) using the optimaldissolved oxygen or carbon dioxide concentration; 4) using inducers andrepressors; 5) varying the above in a time-optimal fashion; 6)minimizing the accumulation of by-products that negatively impact thegrowth or metabolism of the organism, 7) genetically altering theorganism using recombinant DNA or hybridoma technology; and, 8) usingauxotrophic or mutants with altered regulatory systems, 9) and/or thelike. Several methods have emerged to control growth and metabolism in aculture. As the '453 patent illustrates, various techniques useautomated on-line or at-line measurements of the concentration ofgrowth-limiting substrates such as glucose and glutamine. However, ascan be imagined, feedback control based on substrate measurements can berelatively slow and less responsive. Likewise, another method that hasmet some success is to add growth-limiting substrate in an exponentialmanner. However, this exponential growth technique suffers from thedrawbacks of allowing the culture medium to be underfed and/or overfedthereby not obtaining the optimal growth and metabolism characteristics.Another feedback measure of growth and metabolism is to measure thespecific oxygen uptake rate and maintain it at a setpoint correspondingto the desired growth rate. This method is very effective in cultureswith low growth rate. Another method is the dissolved oxygen-stat(DO-stat) method, which will be defined below. Likewise, another methodincludes pH-stat, which will also be defined below. Further methodsinclude carbon dioxide transfer rate measurements, oxygen uptakemeasurements, respiratory quotient (RQ) measurements and the like. Whilethese methods have proven successful under certain conditions, there arepotential limitations associated with each method (see Table 1 forexamples). Accordingly, the art field is in need of an improvedmeasurement for monitoring and controlling the growth and metabolismcharacteristics of a culture.

[0012] The '453 patent discloses a novel method for controlling growthrate and metabolic state in a fed-batch fermentation by measuring thereagent addition rate, pH, oxygen uptake rate, biomass concentration,and reactor volume. The measurement of the reagent addition rate isdivided by the measurement of the oxygen uptake rate and maintained at apre-determined setpoint. Another embodiment is disclosed where a reagentaddition rate is divided by the product of the biomass concentration andthe reactor volume and maintained at a setpoint corresponding to adesired growth rate. A further additional embodiment is disclosedwherein the reagent addition rate and a specific oxygen uptake rate aremaintained at different setpoints corresponding to a desired growthmetabolic rate. However, as can be seen, the process disclosed on the'453 patent requires numerous measurements and calculations, and may bedifficult to implement in commercial production on a routine basis.Therefore, the art field is in need of a process whereby simplemeasurements may be taken without complex calculations resulting inoptimal growth and metabolic characteristics.

[0013] It is common for high cell density microbial fermentations to usea fed-batch mode of operation in order to resolve issues such asmetabolic by-product accumulation or substrate inhibition, equipmentlimitation, etc. Cells are typically grown in batch mode to anintermediate cell density following which feeding of carbon/energyand/or complex nutrients is initiated. The feeding strategies can beclassified into two major categories: (1) open-loop (non-feedback) and(2) closed-loop (feedback) feeding strategies.

[0014] The open-loop feeding strategies are typically pre-determinedfeed profiles for carbon/nutrient addition. There are an infinite numberof feed profiles, but more commonly the feed rates are either constantor increased feed rate (either constant, stepwise or exponential) inorder to keep up with the increasing cell densities. While these simplepre-determined feed profiles have been applied successfully in certaincases, the major drawback is the lack of feed rate adjustment based onmetabolic feedback from the culture. Therefore, the open-loop feedingstrategies can fail if an unexpected disturbance causes the culture todeviate from its “expected” growth pattern.

[0015] The closed-loop feeding strategies, on the other hand, rely on ameasurement that is an indicator of the metabolic state of the culture.Two most commonly measured online variables, the dissolved oxygen (DO)concentration and pH, in microbial fermentation are also key indicatorsof cellular physiology. Therefore, they have traditionally been used asfeedback variables upon which the feed rates are based. These moresophisticated closed-loop feeding strategies, called DO-stat and pH-statwhich are based on the measurement of DO and pH respectively, have beenutilized to minimize accumulation of inhibitory metabolites, such asacetate, during high cell density cultivation^([1,2,3]).

[0016] The traditional DO-stat control of nutrient feeding is simplybased on the concept of DO rises (due to a reduction or cessation ofoxygen consumption or respiration) upon nutrient limitation ordepletion. The DO-stat control maintains the culture at a constant DOlevel (the DO setpoint) by increasing the nutrient feed rate when DOrises above the setpoint and reducing the nutrient feed rate when DOdrops below the setpoint. The DO-stat strategy typically works well indefined media where nutrient depletion results in rapid DO rise.However, the DO-stat method often fails in media supplemented with richcomplex nutrients such as yeast extract, tryptone, peptone, casaminoacid, or Hy-Soy. Rich complex nutrients are capable of supportingcellular maintenance and respiration through amino acid catabolism suchthat the DO level remains low (i.e. no apparent DO spikes) even undercarbon source limitation or depletion.

[0017] When a complex medium is used for culture growth, a ph-statstrategy may be more suitable than DO-stat since the culture pH tends toincrease once the carbon source is depleted. In a manner similar toDO-stat control, the pH-stat method maintains a constant culture pH atabout the setpoint by increasing the nutrient feed rate as pH risesabove the setpoint and reducing the nutrient feed rate when pH dropsbelow the setpoint. However, since the change in culture pH uponnutrient depletion is less responsive than that of DO, feeding controlby pH-stat can be relatively sluggish when compared to DO-stat. Inaddition, the pH-stat control does not work well for culture grown inchemically defined media^([3,4]).

[0018] As explained before, in general, when the carbon source becomeslimiting or depleted in fermentations employing complex medium, theculture pH rises while the DO value remains low. This suggests an activerespiration in the absence of primary carbon source. This pH rise uponcarbon source depletion is due to a combination of metabolism shift (toutilizing complex nitrogen which releases hydroxide after ammoniumuptake) as well as reutilization of excreted acids (such as aceticacid). Similarly, the low DO value during carbon source depletion ismost likely due to the metabolism shift to utilizing amino acids fromthe complex nitrogen feed. The degradation products of amino acids enterthe tricarboxylic acid (TCA) cycle (e.g., 2-oxoglutarate, a deaminatedproduct of glutamate, is an intermediate of the TCA cycle) and maintainactive aerobic respiration, which results in oxygen consumption and lowDO profile even under glucose depletion. Clearly the DO-stat controlwill not function properly if the culture DO concentration remains lowduring glucose limitation.

[0019] Therefore, the art field is in search of an improved method thatcan take advantage of the benefits offered by both pH and DO-statwithout the usual drawbacks.

SUMMARY OF THE INVENTION

[0020] Embodiments of the present invention generally relate tofermentation processes, fermentations, and fermentation compositions. Inan embodiment, novel processes are disclosed for fed-batch andcontinuous fermentation. Generally, technologies of DO-stat and pH-statare combined to control the feed rates of substrate or nutrient (primarycarbon/energy source such as glucose and glycerol), sometimes referredto as nutrient, and enriched oxygen. In an embodiment, the substrate ornutrient (primary carbon/energy source) feed rate is controlled by aDO-stat. In an embodiment, the pH of a fermentation is maintained atabout a setpoint (pH-stat) by adjusting the feed rate of enriched oxygen(which indirectly affect the substrate or nutrient (primarycarbon/energy source) feed rate through DO-stat). In variousembodiments, the pH of a fermentation can also be maintained at about asetpoint by adjusting agitation, aeration, back pressure, or acombination of these components in the fermentor. In further embodiment,a DO-stat and pH-stat are linked. In another embodiment, the linkedDO-stat and pH-stat operate simultaneously to maintain the DO and pH ofa fermentation at about a setpoint by adjusting the feed rate ofsubstrate or nutrient (primary carbon/energy source) and the feed rateof enriched oxygen.

[0021] This summary is not intended to act as a limitation on the scopeof the appended claims. For an understanding of the invention, attentionshould be had on the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

[0022]FIG. 1 is a graph illustrating a plot of a prior art pH-statfermentation feeding control wherein the pH and glucose feed rate of thefermentation are plotted against culture time.

[0023]FIG. 2 is a graph illustrating a plot of the prior art pH-statfermentation feeding control of FIG. 1 wherein the cell growth(expressed as optical density, or OD), glucose consumption and productconcentration of the fermentation are plotted against culture time.

[0024]FIG. 3 is a graph illustrating a plot of pH and enriched oxygenflow rates of a dual DO-pH stat control of an embodiment of the presentinvention plotted against culture time.

[0025]FIG. 4 is a graph illustrating the dissolved oxygen (DO)measurement and the glucose feed rate of a dual DO-pH stat control ofthe embodiment of FIG. 3 plotted against culture time.

[0026]FIG. 5 is an illustration of a graph of cell growth (OD), glucoseconsumption and product concentration of an embodiment of a dual DO-pHstat control of the embodiment of FIG. 3 plotted against culture time.

DETAILED DESCRIPTION OF THE INVENTION

[0027] As used herein, the term “nutrient” means and refers to nutrientsor substrates that may be fed to a fermentation process, such ascarbon/energy sources, such as glucose, carbohydrates, vitamins,minerals, oils, and the like. As used herein, the term “culture” shallmean and refer to at least one medium containing at least one microbialstrain and the like which is suitable for fermentation, such as, but notlimited to, fungal, yeast, and bacterial strains. It is to be understoodthat the term “culture” as used in the present invention includes amedium wherein all necessary components are added to the medium beforethe start of the fermentation process, and further includes a mediumwherein part of the necessary components are added before starting andpart are added to the medium during the fermentation process. As usedherein, the term “dissolved oxygen content” shall mean and refer to thedissolved oxygen content of the medium and/or culture. As used herein,the term “medium” means and refers to a nutrient system for theartificial cultivation of cells, organisms, and/or the like. As usedherein, the term “computer,” means and refers to a programmable machineor other device that can store, retrieve, and process data.

[0028] The type of carbon, nitrogen and complex nitrogen source, whichis used in the rich complex medium, is not critical to the invention.Preferably, a primary carbon source is selected from the groupconsisting of carbohydrates such as glucose, lactose, fructose, sucrose,maltodextrins, starch and inulin, glycerol, vegetable oils,hydrocarbons, alcohols such as methanol and ethanol, organic acids suchas acetate and higher alkanoic acids. More preferably, a carbon sourceis selected from the group consisting of glucose, glycerol, lactose,fructose, sucrose and soybean oil. It is to be understood that the term“glucose” includes glucose syrups, i.e. glucose compositions containingglucose oligomers. Most preferred carbon sources comprise glucose,glycerol, other carbohydrates, and/or other nutrient solutionscontaining one or a combination of these sugars.

[0029] A defined nitrogen source preferably is selected from the groupconsisting of urea, ammonia, nitrate, ammonium salts (such as ammoniumsulphate, ammonium phosphate, ammonium chloride and ammonium nitrate),and amino acids such as glutamate and lysine. More preferably, anitrogen source is selected from the group consisting of ammonia,ammonium sulphate, ammonium chloride and ammonium phosphate. Mostpreferably, the nitrogen source is ammonia. The use of ammonia as anitrogen source has the advantage that ammonia additionally can functionas a pH-controlling agent. In case ammonium sulphate and/or ammoniumphosphate are used as a nitrogen source, at least a portion of thesulphur and/or phosphorus requirement of the microorganism may be met.

[0030] A rich complex nitrogen source preferably is selected from thegroup consisting of one or more of the following components: yeastextract, yeast autolysates, yeast nitrogen base, protein hydrolysates(including, but not limited to, peptones, casein hydrolysates such astryptone and casamino acids), soybean meal, Hy-Soy, tryptic soy broth,cotton seed meal, malt extract, corn steep liquor, molasses, and thelike. More preferably, the complex nitrogen source is selected from thegroup consisting of yeast extract, tryptone, casamino acids, peptone,casein hydrolysate and Hy-Soy. Most preferably, the complex nitrogensource is yeast extract and/or tryptone.

[0031] Accordingly, in varying embodiments, the medium comprises one ormore of the following: glucose, yeast extract, potassium phosphatemonobasic, sodium phosphate dibasic, ammonium sulfate, magnesiumsulfate, calcium chloride, thiamine, kanamycin sulfate, antifoam andtrace elements (including zinc sulfate, ferric chloride, manganesechloride, cupric sulfate, cobalt chloride, sodium molybdate and boricacid).

[0032] In varying embodiments of the present invention, to resolve theproblems associated with DO-stat or pH-stat control strategies inmicrobial fermentations employing complex medium, a new substrate ornutrient (or primary carbon/energy source) feeding strategy wasdeveloped. The new strategy, called the DO-pH dual-stat, utilizes bothDO-stat and pH-stat methodologies simultaneously to control thecarbon/energy source feed via DO-stat by supplementing the culture withenriched oxygen to maintain the culture pH constant at about thesetpoint. As the culture pH rises above the setpoint due tocarbon/energy source (such as glucose) depletion, the supplementaloxygen flow rate is increased (via either manual or automatic control)to elevate the culture DO level. This rise in culture DO results in anincrease in carbon source feed rate (which is under DO-stat control)such that the acidic by-products (as an example, but not limited to,acetate) generated through metabolism of the carbon/energy sourcebalance the generation of ammonium, or other base, from the utilizationof complex nitrogen sources. Thus, the culture pH is maintained at aboutits setpoint by controlling an enriched oxygen feed rate (under pH-statcontrol) that indirectly controls the carbon/energy source feed rate(under DO-stat control). In various embodiments, the enriched oxygenfeed (that is under pH-stat control) may be substituted by agitation,aeration, fermentor back pressure, or a combination of these components.In another embodiment, the carbon/energy source (such as glucose) feedrate is proportional to the dissolved oxygen content in thefermentation. In further embodiment, this unique DO-pH dual-statstrategy provides a well-balanced nutrient feed control. In other words,the dual-stat's feed-on-demand approach, which is based on the feedbackof culture pH and dissolved oxygen, essentially eliminates (orminimizes) the typical drawbacks encountered in other types of feedingstrategies, such as excessive by-product accumulation and reduced growthrate due to overfeeding or underfeeding, thereby enhancing theefficiency of the fermentation. An additional advantage of the presentinvention is that it does not require any at-line or off-linemeasurement of growth-limiting substrates or metabolites. It is based onthe feedback of real-time DO and pH on-line measurements throughstandard DO and pH probes.

[0033] In general, processes of the present invention comprise the stepof maintaining a pH of a fermentation medium at about a setpoint bycontrolling oxygen feed, and/or agitation, aeration, back pressure, or acombination of these components. In varying embodiments, controllingoxygen feed (and/or agitation, aeration, back pressure, or a combinationof these components) indirectly controls a nutrient feed rate(carbon/energy source feed rate). Further, the oxygen feed may have anycontent/percentage oxygen, such as air, enriched oxygen, pure oxygen,and/or any concentration thereof. The oxygen feed to the fermentationmay be from one source and/or inlet or through multiple sources and/orinlets. Oxygen feed may be controlled by manners common in the art, suchas by controlling the oxygen feed rate.

[0034] Processes of the present invention may be used in anyfermentation or cell culture. In preferred embodiments, the fermentationis a fed-batch, continuous, and/or combination of these employing richcomplex nutrients.

[0035] In various embodiments, the oxygen feed rate (and/or agitation,aeration, back pressure, or a combination of these components) of thefermentation is controlled by a pH-stat. In further embodiment, the feedrate of carbon/energy source is about proportional to the dissolvedoxygen content (i.e., DO-stat). However, in other embodiments, theenriched oxygen feed can also be controlled manually (instead of underpH-stat control). For example, a constant oxygen feed rate may be usedas long as the enriched oxygen flow rate is maintained at a sufficientlevel such that the glucose feed rate (under DO-stat control) is highenough to prevent culture pH from rising. A rise in culture pH(excluding the case of base addition) typically relates to metabolismshift from primary carbon/energy source to complex nitrogen (or othersecondary carbon/energy sources such as amino acids) due to underfeedingof primary carbon/energy source(s). In other embodiments, the primarycarbon/energy source (e.g., glucose) feed rate may be other thanproportional and may have no comparable relation to the dissolved oxygencontent.

[0036] In various embodiments, the fermentation can be manuallycontrolled or automated by a computer or other machine. In anembodiment, the pH is monitored on a computer. In further embodiment, acomputer and/or computer aided device controls and/or adjusts an oxygenfeed rate (and/or agitation, aeration, back pressure, or a combinationof these components) to maintain the pH at about a setpoint. Furtherembodiments monitor the dissolved oxygen content with a computer and/orcomputer aided device. The use of computer in this application isspecifically intended to include all necessary programs, controllers,devices and/or the like to function as claimed and/or described.

[0037] In certain preferred embodiments, the computer or the computeraided device or other computer or computer aided device controls thesubstrate or nutrient (primary carbon/energy source) feed rate inrelation to the dissolved oxygen content, such as with a DO-stat. In amost preferred embodiment, the substrate or nutrient (primarycarbon/energy source) feed rate is proportional to the error between theDO content reading and the DO setpoint, and is controlled to maintainthe DO level at about a setpoint.

[0038] Accordingly, the present invention comprises the steps of:maintaining a pH of a fermentation medium at about a setpoint bycontrolling an oxygen feed rate which indirectly controls a nutrientfeed rate. In an embodiment, the fermentation process is fed-batchfermentation. In other embodiments, the fermentation process iscontinuous fermentation.

[0039] Embodiments of the present invention also envision an apparatusand/or fermentor. In various embodiments, the fermentation comprises afermentor, multiple nutrient feeds including substrate or nutrient(primary carbon/energy source such as glucose) feed and rich complexnitrogen feed (such as yeast extract solution), a fermentation batchmedium, base, air and oxygen feed. The pH of the fermentation ismaintained at about a setpoint by adjusting the oxygen feed rate (and/oragitation, aeration, back pressure, or a combination of thesecomponents), which indirectly controls the feed rate of substrate ornutrient (primary carbon/energy source) to the medium. Furtherembodiments comprise a pH-stat and/or a DO-stat, whereby the oxygen feedrate (and/or agitation, aeration, back pressure, or a combination ofthese components) is controlled by the pH-stat and the substrate ornutrient (primary carbon/energy source) feed rate is controlled by theDO-stat.

[0040] In another embodiment, the present invention envisions afermentation apparatus comprising: a fermentor, a nutrient feed, a richcomplex nitrogen feed, a fermentation batch medium, a base feed, an airfeed, and an oxygen feed wherein the pH of the fermentation ismaintained at about a setpoint by controlling an oxygen feed rate whichindirectly controls a nutrient feed rate to a culture. The apparatus maybe modified, in an embodiment, such that the fermentation apparatusfurther comprises a pH-stat, whereby at least one of the oxygen feedrate, agitation, aeration, fermentor back pressure, or a combination ofthese components is controlled by the pH-stat. Further embodimentscomprise a DO-stat, whereby the nutrient feed rate is controlled by theDO-stat. Other embodiments further comprise a computer, whereby thecomputer controls the pH-stat and/or the DO-stat.

EXAMPLES

[0041] Table 1 demonstrate a general performances of the DO-pH dual-statwhen compared to the performance of a pH-stat, open-loop and DO-statcontrol for a recombinant E. coli fermentation, the process for which iswell known in the art. TABLE 1 Comparison of different feedingstrategies in fermentation Feeding DO-pH strategy Open-loop DO-statpH-stat Dual-stat Defined + + − + Media (fast (slow response) (fastresponse) response) Rich + − +/− + Complex (may not (slower (fast Mediawork) response; response) metabolic shift) Metabolic no yes Yes YesFeedback (predetermined)

[0042] As can be seen, when compared to the traditional feedingstrategies described earlier (open-loop, standard DO-stat, and pH-stat),features of the dual-stat strategy are:

[0043] 1. The new DO-pH dual-stat feeding strategy works well inculture(s) supplemented with rich complex nutrients while the DO-statmay not function well, and that pH-stat alone shows typical slowresponse;

[0044] 2. The new DO-pH dual-stat feeding strategy will also work wellfor culture(s) grown in defined media whereas a pH-stat may not;

[0045] 3. The new DO-pH dual-stat feeding strategy retains theadvantages of fast responsiveness and metabolic feedback control in bothdefined media and complex media;

[0046] 4. The new DO-pH dual-stat control strategy does not require anyat-line or offline measurement of growth-limiting substrates ormetabolites. It is based on the feedback of real-time DO and pH on-linemeasurements through standard DO and pH probes;

[0047] 5. The new DO-pH dual-stat feeding methodology can also beapplied to non-microbial cultivation(s) such as animal or insect cellculture(s).

[0048] The data illustrated in the figures have been derived fromfermentation runs with a pH-stat and a DO-pH dual-stat, as indicated bythe figure.

[0049] Experimental conditions of the following runs were as follows:

[0050] A 2.8-L shake flask containing 1,000±50 mL of LB seed medium(with 30±5 mg/L kanamycin sulfate) was inoculated with 50±2 μL stockculture from a Development Cell Bank vial (Escherichia coli strainBLR(DE3) expressing a recombinant protein). The seed flask was incubatedat 37.0±1.0° C., 200±20 RPM until the OD₆₀₀ reached 1 to 4. This culturewas used to inoculate a 15-L production fermentor (10-L typical workingvolume) containing 8.0±0.5 L production medium supplemented with 30±5mg/L kanamycin sulfate.

[0051] The production medium contained the following components:glucose, yeast extract, potassium phosphate monobasic, sodium phosphatedibasic, ammonium sulfate, magnesium sulfate, calcium chloride,thiamine, kanamycin sulfate, antifoam and trace elements (including zincsulfate, ferric chloride, manganese chloride, cupric sulfate, cobaltchloride, sodium molybdate and boric acid).

[0052] The fermentation process was controlled at 37.0±1.0° C. and pH7.0±0.3 (with ammonia hydroxide) for about 9 to 12 h. Airflow was keptconstant at 8.0±1.0 SLPM throughout. The dissolved oxygen (DO) wasmaintained at about 30% saturation (preferably above 10% saturation)either by a constant agitation at 1000 RPM throughout; or bycontinuously increasing the agitation rate from 300 to 1,000 RPM(variable agitation mode). In the variable agitation mode, upon reachingthe maximum agitation rate (1,000 RPM) the agitation control wasswitched to a manual constant 1,000 RPM for the rest of cultivation. Anyfurther demand for dissolved oxygen was met by supplementary enrichedoxygen in the range of 0.0 to 5.0 SLPM. When the OD₆₀₀ was 8 to 15, a20% (w/v) yeast extract solution was fed to the fermentor at 2.0±0.5g/min constant rate. When the residual glucose concentration wasdepleted (as indicated by a pH rise as well as glucose analyzerreading), a 50% (w/v) glucose feed, under the DO-pH dual-stat control(DO setpoint at 30% saturation), was activated. When the OD₆₀₀ reached30 to 40 (about 5 to 8 hours elapsed fermentation time), the culture wasinduced for recombinant protein production by adding Isopropylβ-D-1-thiogalactopyranoside (IPTG) to a final concentration between 10and 15 mM.

[0053] Three to four hours after induction, with an OD₆₀₀ 50 to 70, thefermentation culture was ready for cooling and subsequent harvest. Totalfermentation time was typically 9 to 12 hours.

[0054] Now referring to FIG. 1, under the prior art pH-stat controlscheme, as the pH of the fermentation increased above the setpoint (pH7.0), the glucose feed rate increased. However, due to a slower pHresponse, the glucose feed rate was shown to be oscillating. Oscillationin pH and glucose feed rate results in a cycling of overfeeding andunderfeeding of glucose to the culture, which leads to suboptimal growthconditions, lower product yield and/or increased operating costs.

[0055] It can be seen in FIG. 2 that between h 6 and h 8 elapsed time,the rates of cell growth and product formation decreased as a result ofreduced glucose feed. Under the condition of glucose underfeeding inpH-stat control, the final product yield was only half of that in adual-stat control process (see FIG. 5). Likewise, this process is notoptimized.

[0056] In contrast, the pH and oxygen feed rate in a DO-pH dual-statfermentation did not exhibit any oscillation (shown in FIG. 3), nor didthe glucose feed rate (see FIG. 4). Under DO-pH dual-stat control, theglucose feed rate during induction phase (between approximately h 6 andh 9 elapsed time) was more than twice (about 2 mL/min; FIG. 4) of thatunder pH-stat control (less than 1 mL/min; see FIG. 1). In both pH-statand DO-pH dual-stat processes, the glucose concentration in the cultureremained below about 0.1 g/L throughout the entire feeding period (datanot shown).

[0057] Now referring to FIG. 5, it can be observed that measurements ofembodiments of the present invention produce optimized cell growth andproduct concentration. A comparison of FIG. 2 and FIG. 5 illustratesthat an embodiment of a DO-pH dual-stat of FIG. 5 allows steady cellgrowth and dramatically increases the same product concentration. As canbe seen, in FIG. 5, product concentration was maximized at the end ofthe run to above 3.0 g/L. FIG. 2 maximized product concentration at theend of the run to about 1.5 g/L. Therefore, the novel process of thepresent invention nearly about doubled final product concentration inthe compared runs.

[0058] While the invention has been described in connection withspecific embodiments thereof, it will be understood that it is capableof further modifications and this application is intended to cover anyvariations, uses, or adaptations of the invention following, in general,the principles of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims. Such further and other embodiments arecontemplated whereby additions of an acid and/or a base may be made tothe fermentation. Further, all patents mentioned herein are herebyincorporated by reference.

REFERENCES

[0059] 1. Mori, H., Yano., T., Kobayashi, T. and Shimizu, S. (1979).High density cultivation of biomass in fed-batch system with DO-stat.Journal of Chemical Engineering of Japan, 12(4), 313-319;

[0060] 2. Konstantinov, K., Kishimoto, M., Seki, T. and Yoshida, T.(1990). A balanced DO-stat and its application to the control of aceticacid excretion by recombinant Escherichia coli. Biotechnology andBioengineering, 36, 750-758.

[0061] 3. Suzuki, T., Yamane, T. and Shimizu, S. (1990).Phenomenological background and some preliminary trials of automatedsubstrate supply in pH-stat modal fed-batch culture using a setpoint ofhigh limit. Journal of Fermentation and Bioengineering, 69(5), 292-297.

[0062] 4. Lee, S. Y. (1996). High cell-density culture of Escherichiacoli. Trends in Biotechnology, 14, 98-105.

[0063] 5. Wong, H. H., Kim, Y. C., Lee, S. Y. and Chang, H. N. (1998).Effect of post-induction nutrient feeding strategies on the productionof bioadhesive protein in Escherichia coli. Biotechnology andBioengineering, 60, 271-276.

What is claimed is:
 1. A process for conducting fermentation comprisingthe step of: maintaining a pH of a fermentation medium at about asetpoint by controlling an oxygen feed rate which indirectly controls anutrient feed rate.
 2. The process of claim 1 wherein the fermentationprocess is fed-batch fermentation.
 3. The process of claim 1 wherein thefermentation process is continuous fermentation.
 4. The process of claim1 further comprising adding a rich complex nitrogen feed to thefermentation medium.
 5. The process of claim 1 further comprising addinga base.
 6. The process of claim 1 further comprising adding air and/orenriched oxygen feed.
 7. The process of claim 1 wherein the oxygen feedrate of the fermentation is controlled by a pH-stat.
 8. The process ofclaim 1 wherein the nutrient feed rate is proportional to a dissolvedoxygen (DO) content in the fermentation.
 9. The process of claim 1wherein the nutrient is selected from the group consisting of glucose,glycerol, other carbohydrates, and other nutrient solutions containingone or a combination of these sugars.
 10. The process of claim 1 furthercomprising monitoring the pH of the fermentation on a computer.
 11. Theprocess of claim 10 wherein the computer performs the step ofcontrolling the oxygen feed rate.
 12. The process of claim 8 wherein thenutrient feed is controlled by a DO-stat whereby the nutrient feed rateincreases when the culture dissolved oxygen is above its setpoint ordecreases as the dissolved oxygen is below its setpoint.
 13. The processof claim 11 wherein a ph-stat controls the oxygen feed rate, agitation,aeration, fermentor back pressure, or a combination of these components.14. The process of claim 11 wherein the computer controls the nutrientfeed rate.
 15. The process of claim 1 wherein the medium is selectedfrom the group consisting of a defined medium, semi-defined medium, anda rich complex medium.
 16. The process of claim 1 whereby the oxygenfeed rate is constant at a rate sufficient to prevent culture pH fromrising.
 17. The process of claim 8 nutrient feed rate is proportional tothe error between the DO content and the DO setpoint.
 18. A fermentationapparatus comprising a fermentor, a nutrient feed, a rich complexnitrogen feed, a fermentation batch medium, a base feed, an air feed,and an oxygen feed wherein the pH of the fermentation is maintained atabout a setpoint by controlling an oxygen feed rate which indirectlycontrols a nutrient feed rate to a culture.
 19. The fermentationapparatus of claim 17 further comprising a pH-stat, whereby at least oneof the oxygen feed rate, agitation, aeration, fermentor back pressure,or a combination of these components is controlled by the pH-stat. 20.The fermentation apparatus of claim 17 further comprising a DO-stat,whereby the nutrient feed rate is controlled by the DO-stat.
 21. Thefermentation apparatus of claim 17 further comprising a computer,whereby the computer controls the pH-stat and/or the DO-stat.